人工智慧影像探討ROCK抑制劑對角膜細胞之影響

Abstract

角膜傷口癒合涉及細胞遷移、傷口覆蓋、增殖與細胞骨架重塑等多種細胞行為。Rho相關捲曲螺旋蛋白激酶（Rho-associated coiled-coil-containing protein kinase, ROCK）訊號路徑與細胞骨架張力、細胞黏附及遷移調控密切相關，而ROCK抑制劑（ROCK inhibitor）已被廣泛探討於角膜內皮細胞再生、內皮損傷修復與角膜內皮疾病治療中，並被認為具有促進角膜傷口癒合之潛力。由於ROCK inhibitor若作為潛在點眼治療策略，藥物可能經由角膜上皮層進入並於基質層中擴散，因此在評估其作為角膜損傷治療藥物之可行性時，除了關注其對角膜內皮細胞的促進作用外，亦需進一步釐清其對角膜基質細胞行為之影響。我們結合連續時序（time-lapse）影像並運用AI輔助細胞形態學分析接著利用免疫螢光染色驗證，評估藥物處理後細胞在傷口癒合過程中的行為與分子變化。過程中，我們建立體外傷口癒合模型，觀察細胞於0 - 12小時內的動態修復過程，並分析細胞修復行為的差異；同時利用無標記影像進行單細胞層級形態分析觀察細胞型態差異，並以免疫螢光染色探討相關蛋白分布結果。結果表示，Y-27632促進角膜基質細胞傷口區域之閉合趨勢，並非單純來自細胞遷移速度或細胞數量的增加，而可能與細胞移動方向性、空間分布、細胞型態有關。形態學與免疫螢光染色結果說明，Y-27632會改變細胞鋪展狀態與相關蛋白分布，且α-SMA未見明顯陽性訊號，表示藥物未明顯誘導角膜基質細胞朝肌成纖維細胞表型轉化。有絲分裂持續時間分析則顯示，藥物濃度相同下對角膜內皮細胞具有顯著影響，但於角膜基質細胞中未達顯著差異，推測其作用可能具有細胞類型差異。綜觀所述，我們推測Y-27632於角膜基質細胞傷口癒合中，可能調控細胞骨架排列、細胞鋪展狀態、遷移方向性與傷口覆蓋能力。在未明顯誘導肌成纖維細胞分化的情況下促進修復，並可能具有降低瘢痕化相關反應之潛力。

During corneal wound healing, cells undergo coordinated migration, wound coverage, proliferation, and cytoskeletal remodeling. The Rho-associated coiled-coil-containing protein kinase (ROCK) pathway regulates actomyosin contractility, cell adhesion, and migration, and ROCK inhibitors have been studied as potential agents for corneal endothelial regeneration and repair. However, their effects on corneal stromal cells remain less defined, although stromal cells may also be exposed to the drug during topical application. In this study, we investigated how Y-27632 regulates repair-associated behaviors in corneal endothelial and stromal cells. Using an in vitro wound model with time-lapse imaging, AI-assisted segmentation, single-cell analysis, label-free imaging, and immunofluorescence staining, we monitored cellular and protein-distribution changes during the first 12 hours after wound creation. Y-27632 promoted a trend toward wound gap closure in corneal stromal cells, possibly by modulating migration directionality, spatial distribution, cell spreading, and cytoskeletal organization. Label-free imaging and TOMM20 staining further suggested broader mitochondrial distribution after ROCK inhibition. No obvious α-SMA-positive signal was observed, indicating no marked induction of myofibroblast-like differentiation. Mitotic duration was significantly affected in corneal endothelial cells but not stromal cells, suggesting a cell-type-dependent response. Together, these findings suggest that Y-27632 may support corneal stromal wound healing while potentially limiting fibrosis-related responses.